1. Field of the Invention
The present invention relates to a new maltase enzyme, its preparation and use in amylase assay procedures.
2. Description of the Prior Art
Prior to the present invention it has been considered that a single strain of the yeast Saccharomyces would produce only a single maltase enzyme Halvorson et al. Biochimica et Biophysica Acta, volume 67, pages 542-553, prepared maltase enzymes from five different strains of the yeast Saccharomyces. Each of these strains produced what appeared to be the same species of maltase (.alpha.-glucosidase), since the maltase produced by the process is indistinguishable with respect to heat of inactivation, electrophoretic mobility, chromotography from either CM-cellulose or DEAE cellulose columns, neutralization with specific antiserum and, most importantly, substrate specificity.
One use for maltase has been found to be in testing enzymes for amylase. Amylase assay involves measuring the rate of which the amylase catalyzes the hydrolysis of a substrate into glucose. Typically the measurement is accomplished by use of the maltase enzyme, to convert maltose into glucose. The glucose is then converted to glucose-6-phosphate by reaction with ATP in the presence of the hexokinase enzyme. The glucose-6-phosphate is oxidized in the presence of the glucose-6-phosphate dehydrogenase (Leuconostoc) enzyme to 6-phosphogluconate in the presence of NAD (nicotinamide adenine dinucleotide) which is reduced to NADH (reduced from NAD). The conversion of NAD to NADH changes the absorption of the solution at 340nm. By measuring the change in the absorption, it is possible to measure reaction rate. Diagramatically, using oligosaccharide as the substrate, the process comprises:
__________________________________________________________________________ (1) Oligosaccharide ##STR1## maltose + maltotriose +smaller oligosaccharides (2) maltose + maltotriose ##STR2## glucose (3) glucose + ATP ##STR3## glucose-6- + ADP phosphate (4) glucose-6-phos- ##STR4## 6-phospho- phate + NAD gluconate + NADH __________________________________________________________________________
The problem encountered in using this amylase assay technique is that previously known maltase enzymes also catalyze the hydrolysis of oligosaccharides to maltose. This reaction was indicative that even when no amylase was present, NAD was, never the less, being converted to NADH which changed the absorption of the solution. The rate of this reaction is called the blank rate. The blank rate must be determined before beginning the amylase assay test and must be accounted for before one can determine how much amylase is present from the reaction kinetics. Since the blank rate introduces a source of potential error, the amylase assay solutions are prepared in a manner such that the blank rate is minimized. This minimization of the blank rate reduces the overall sensitivity of the amylase assay technique.
Accordingly, there exists a need for a method of increasing the sensitivity of amylase assay techniques while simultaneously reducing the blank rate.